Compositions for inhibiting atherosclerosis

ABSTRACT

The present invention relates to compositions and methods for the reduction of atherosclerotic plaques and the decrease in the level of total serum cholesterol, triglycerides, serum LDL cholesterol, and serum HDL cholesterol.

PRIORITY CLAIM

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/952,003, filed Sep. 28, 2004, which is acontinuation-in-part of International Patent Application No.PCT/BR03/0049, filed Mar. 28, 2003 and published in English on Oct. 9,2003 as WO 03/082324 (priority to both Ser. No. 10/952,003 andPCT/BR03/0049 being claimed herein), and further claims priority to U.S.Provisional Application Ser. No. 60/890,977, filed Feb. 21, 2007, allthree of which are incorporated by reference in their entireties herein.

1. INTRODUCTION

The present invention relates to compositions and methods for thereduction of atherosclerotic plaques and the decrease in the level oftotal serum cholesterol, triglycerides, serum LDL cholesterol, and serumHDL cholesterol.

2. BACKGROUND

Current treatment for atherosclerosis involves lipid-loweringmedications and drugs that affect lipid metabolism, including statins,bile acid absorption inhibitors, cholesterol absorption inhibitors,fibrates and antioxidants such as probucol, among others. (Zipes et al.Eds., 2005, Braunwald's Heart Disease, Elsevier Saunders, Philadelphia).These treatment regimens are based, at least in part, on the theory thatoxidized lipoproteins are the main causative factor of atherosclerosis.However, the exact mechanism by which cholesterol oxidizes is still notfully understood.

Archaea are the most ancient microorganisms existing in nature, but havebeen characterized only recently. See, Woese et al., Proc Natl. Acad.Sci. U.S.A. 74: 5088-5090 (1977). They inhabit extreme environments andare constituted by lipid monolayer membranes. Rich alkaline atmospherewith sodium ions and metals prevents proliferation of other bacteria,but is favorable to archaea's growth. Archaea have been isolated fromalkaline waters from the Dead Sea, the Great Salt Lake and YellowstoneNational Park. They have a small size, can—just barely—be viewed with anoptical microscope, and observation of structural details requireselectron microscopy. See, Howland et al., The surprising archaea.Discovering another domain of life, Oxford University Press (New York,2000). Some are considered hyperthermophilic as they survive in veryhigh temperatures.

Another unusual characteristic of some archaea is that they appear touse metal as an energy source. See, Amend et al., F.E.M.S. Microbiol.Rev. 25: 175-243 (2001). It is considered that archaea usually need ananaerobic or nearly anaerobic environments to carry outoxidation-reduction reactions with participation of different chemicalcompounds, including metals.

Recently, a new kind of extremely small archaea, which is dependent onbigger archaea, was described and named nanoarchaea. See, Huber J etal., Nature 417: 63-67 (2002). With the exception of archaea that residein the mammalian intestine and produce methane gases, there is no reportof archaea existing within plants or animals. See, Florin T H J et al.,Am. J. Gastroenterol. 95: 2872-2879 (2000).

3. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for thereduction of atherosclerotic plaques. Without being limited by theory,it is based on the hypothesis that the presence of mycoplasma and one ormore other microorganism promotes atheroma formation. The compositionsand methods of the invention may also be used to decrease the level oftotal serum cholesterol, triglycerides, serum LDL cholesterol, and serumHDL cholesterol. In one non-limiting embodiment of the invention, thecomposition comprises an agent that removes sialic acid residues, ametal chelator, and optionally one or more purified plant extracts.

In a preferred non-limiting embodiment of the invention, the compositioncomprises a protein capable of removing sialic acid residues, such as aneuraminidase enzyme and/or a trans-sialidase enzyme; a metal chelator,preferably pyrrolidine dithiocarbamate (PDTC), along with one or morepurified plant extracts. The purified plant extract may be derived froma plant selected from the group consisting of Allium sativum (garlic),Ginkgo biloba, tomato, orchids of the genus Cymbidium and Dendrobium,for example, Cymbidium ssp, Dendrobium nobile and Dendrobium moschatum;guava, ginseng, for example, Pfaffia paniculata (Brazilian ginseng);Zingiber officinale (ginger), and tobacco, wherein the purified extractcomprises particles containing DNA or RNA, such as an archaea or ananoarchaea.

The present invention also provides methods for increasing the number ofnon-pathogenic archaea in a plant extract, while also decreasing thenumber of pathogenic archaea in the plant extract. In one embodiment,the non-pathogenic archaea in a plant extract are increased and thepathogenic archaea in the plant extract are decreased by aging the plantextract, and then diluting the plant extract with thermal water,followed by an additional aging period.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-J. (A-E) shows macroscopic aortic atheroma plaques (arrows) and(F-J) shows Chlamydia pneumoniae positive antigen expression in aortalintimal areas (arrows) of rabbits fed a 1% cholesterol diet andsubmitted to different anti-atherosclerotic treatments. Group II (A, F)received no treatment, Group III (B, G) was treated with trans-sialidase(“TS”)+pyrrolidine dithiocarbamate (“PDTC”), Group IV (C, H) was treatedwith TS+PDTC+Allium sativum (“AS”), Group V (D, I) was treated withTS+PDTC+AS+Ginko biloba (“GB”), and Group VI (E, J) was treated withTS+PDTC+AS+GB+Zingiber officinale (“ZO”).

FIG. 2 shows the nucleotide sequence of a plasmid encoding the catalytictrans-sialidase unit of trans-sialidase from Trypanosoma cruzi (SEQ IDNO:3). The letters in capital represent the pET14b plasmid and theunderlined letters correspond to the position of the oligonucleotidesused to amplify the Trypanosoma cruzi clone.

FIG. 3 shows the amino acid sequence of the protein encoded by thenucleic acid sequence depicted in FIG. 2. (SEQ ID NO:4). In bold are theamino acids not found in the original clone.

FIG. 4 shows small dark electron-dense nanoarchaea of between 0.03-0.15μm in diameter.

FIG. 5 shows dark medium sized electron-dense archaea of between 0.5-1.1μm in diameter, and large clear, empty archaea of between 1.0-2.4 μm indiameter.

FIG. 6 shows clear, empty archaea of between 0.15-2.0 μm in diameter.

FIG. 7 shows an electron micrograph of a human aortic aneurysm. Theaortic aneurysm exhibits many round lipidic bodies in both the cytoplasmof macrophages and in the extracellular matrix. The round lipidic bodiesare surrounded by immunogenic lymphocytes.

FIG. 8 shows a high magnification view of the round lipidic bodydescribed in FIG. 7. The round lipidic body exhibits a clear externalmembrane corresponding to the morphology of the large lipidic archaeamicrobes shown in FIGS. 5 and 6 (and also isolated from tobacco).

5. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity, and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

(i) compositions for treating atherosclerosis; and

(ii) therapeutic uses.

5.1 Compositions for Treating Atherosclerosis

The present invention provides for compositions and methods that preventor treat diseases associated with undesirable cell proliferation andfibrosis. Specifically, the compositions and methods of the inventioninhibit the narrowing of blood vessels and reduce atherosclerosis. Thecompositions and methods of the present invention also decrease thelevel of total serum cholesterol as well as serum LDL, serum HDL andtriglyceride levels in a treated patient.

In particular embodiments of the invention, the composition comprises aprotein capable of removing sialic acid residues, wherein removal of thesialic acid residues inhibits or prevents the attachment of a mycoplasmaand one or more non-mycoplasma microorganism to a host cell. Preferrednon-limiting embodiments further comprise a metal chelator and/or one ormore purified plant extracts. Administration of the compounds of theinvention has the effect of reducing the presence of atheroscleroticplaques on a blood vessel, and decreasing the level of blood serumlipids, total serum cholesterol, serum LDL, serum HDL, and triglyceridesof a treated individual.

The term “composition” as used herein means agents or mixtures orcombinations thereof effective to prevent or reduce the rate of growthof an atherosclerotic lesion and/or to decrease the presence of amycoplasma and non-mycoplasma microorganism with an atheroscleroticplaque. In one embodiment, the composition inhibits the ability of themycoplasma and non-mycoplasma to associate with a substrate, forexample, but not limited to, a blood vessel. In another non-limitingembodiment, the composition inhibits the association of a mycoplasma anda non-mycoplasma microorganism.

The term “atherosclerosis,” “atherosclerotic plaque,” “plaque,” or“atheroma” as used herein refers to the accumulation of one or more oflipids, cholesterol, collagen, and macrophages on the walls of asubject's blood vessel. The presence of plaques in a blood vessel canalso be associated with ossification and calcification of the bloodvessel walls.

The term “blood serum lipids” as used herein refers to HDL and LDLlipoproteins.

The term “HDL” as used herein means high density lipoprotein.

The term “LDL” as used herein means low density lipoprotein.

In further non-limiting embodiments of the invention, the mycoplasma maybe Mycoplasma (M.) buccale, M. faucium, M. fermentans, M. genitalium, M.hominis, M. lipophilum, M. oral, M. penetrans, M. pneumoniae, M.salivarium, or M. spermatophilum, wherein the mycoplasma is associatedwith one or more additional non-mycoplasma microorganisms. The one ormore additional non-mycoplasma microorganism may be a bacteria, archaeaor virus, for example, but not limited to, spirochete or chlamydia suchas Chlamydia pneumoniae. According to the invention, the mycoplasma andnon-mycoplasma may be attached to a substrate, for example, but notlimited to, a blood vessel or an atherosclerotic plaque. In a furthernon-limiting embodiment, the mycoplasma and non-mycoplasma are attachedto the substrate by sialic acid.

In a preferred embodiment of the invention, the protein capable ofremoving sialic acid residues is a trans-sialidase or neuraminidaseenzyme A combination of such enzymes or an enzyme having both activitiesmay also be used.

In certain non-limiting embodiments, the composition comprises aneuraminidase enzyme of, for example but not limited to, Bacteroidesfragilis, Streptococcus pneumoniae, Streptococcus oralis, Arthrobacterureafaciens, Clostridium perfringens, Mycoplasma alligatoris,Arcanobacterium pyogenes, Clostridium sordellii, Pseudomonas aeruginosa,Micromonospora viridifaciens, Vibrio cholerae. Streptomyces avermitilis,Influenza virus, Streptomyces coelicolor, Flavobacteriales bacterium,and Solibacter usitatus.

In other non limiting embodiments, the protein is a trans-sialidase, forexample, the trans-sialidase enzyme of Trypanosoma brucei.

In a preferred embodiment, the composition is the trans-sialidase enzymeof Trypanosoma cruzi, or a portion or variant of the native enzyme whichhas trans-sialidase activity.

Alternatively, the trans-sialidase enzyme can be a recombinanttrans-sialidase enzyme.

In specific non-limiting embodiments, the recombinant trans-sialidase isas described in International Patent Publication WO 2002/002050 byHiguchi et al., published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 byHiguchi et al., issued Sep. 19, 2006. For example, the trans-sialidasegene may be obtained from a genomic clone, isolated from a commerciallyavailable lambda Zap®II library (Stratagene, http://www.stratagene.com)of T. cruzi Y strain (Silva and Nussenzweig, 1953, Folia Clin Biol 20:191-203), as described in Uemura et al. (Uemura et al., 1992, EMBO J 11:3837-3844). From the original lambda clone, which expresses enzymaticactivity, an SK plasmid containing the trans-sialidase gene may begenerated (SK-154-0). The preferred plasmid used is pTSII, whichcorresponds to a fragment of the original gene (clone 154-0) amplifiedthrough PCR, and inserted into the sites Ndel and BamHl of the vectorpET14b (Novagen—http://www.novagen.com). The PCR product may beamplified using SK-154-0 as a template with the following primers:

(SEQ ID NO:1) a) TSPET14: 5′-GGAATTCCATATGGCACCCGGATCGAGC (SEQ ID NO:2)b) RT154: 5′-CGGATCCGGGCGTACTTCTTTCACTGGTGCCGGT

The resulting PCR product should have a nucleic acid sequence as setforth in FIG. 2 (SEQ ID NO:3), and a corresponding amino acid sequenceas depicted in FIG. 3 (SEQ ID NO:4). The resulting plasmid may betransformed into the Escherichia coli BLB21 DE3. The construct can bemade in two steps due to an internal BamHl site in the trans-sialidasegene. The PCR product may be treated with BamHl and Ndel enzymes, andthe resulting fragments fractionated by electrophoresis on an agarosegel. The separated fractions may then be purified from the gel with theSephaglass purification kit (Amersham-Pharmacia). The 5′ Nde1-BamH1digestion fragment may be ligated into the pET14b vector which has beenpre-digested with BamHl and Ndel. The ligation products may be used totransform K12 DH5a E. Coli cells. The plasmid containing E. coli cellsmay be selected and the plasmid purified by methods known in the art.The purified construct may be treated with BamH1, shrimp alkalinephosphatase, and ligated with the BamHI-BamHI-3′ fragment purified fromthe fractionation gel. The ligation products may then be used totransform K12 DH5a E. coli cells, from which clones expression oftrans-sialidase may be selected and purified. The final plasmid may beconfirmed by restriction analysis and used to transform the BLB21 DE3pLys strain of E. coli, from which recombinant trans-sialidase enzymecan be purified, as described in International Patent PublicationWO/2002/002050 by Higuchi et al., published Jan. 10, 2002; and U.S. Pat.No. 7,108,851 by Higuchi et al., issued Sep. 19, 2006.

Alternatively, the trans-sialidase enzyme may be purified from a cultureof Trypanosoma cruzi, such as, for example, a culture according toKloetzel et al. (Kloetzel et al., 1984, Rev. Inst. Med. Trop. SaoPaulo., 26:179-85). Supernatant from the culture may be filtered througha 1 μm pore filter in a vacuum chamber. The enzyme may be furtherpurified by filtering the supernatant through a 0.22 μm filter and thenprecipitating the filtrate with a 50% (NH₄)₂SO₄ solution. Theprecipitates may then be dialyzed against phosphate-buffered saline, andpassed through a tresyl-agarose column comprising an immobilizedanti-trans-sialidase monoclonal or polyclonal antibody. The column maybe washed with phosphate-buffered saline, followed by an additional washwith 10 mM sodium phosphate, pH 6.5. The trans-sialidase may then beeluted with a 3.5 mM MgCl₂, 10 mM sodium phosphate, pH 6.0 solution. Thefractions eluted from the column may be filtered through a Sephadex G-25column equilibrated with 20 mM Tris-HCl, pH 8.0, to remove the MgCl₂.The trans-sialidase may be further purified by passage through a Mono Qcolumn equilibrated in 20 mM Tris-HCl, pH 8.0, and eluted with a lineargradient from 0 to 1 mM NaCl in the same buffer.

The purified enzyme derived from the culture should comprise 400 kDamultimeric aggregates. The enzymatic activity of the purifiedtrans-sialidase may be measured according to methods described inInternational Patent Publication WO 2002/002050 by Higuchi et al.,published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 by Higuchi et al.,issued Sep. 19, 2006.

In non-limiting embodiments, the purified trans-sialidase has anenzymatic activity of between 0.1 and 10 U/ml, more preferably between1.0 and 5.0 U/ml, and most preferably 1.3 U/ml.

In certain non-limiting embodiments, the composition comprises a metalchelator, for example, but not limited to, Nitrilotriacetate (NTA),diphenylthiocarbazone(dithizone), histidine, the lipophilic metalchelator DP-109, ethylene glycol tetraacetic acid (EGTA),ethylenediaminetetraacetic acid (EDTA), DMPS(2,3-dimercapto-1-propanesulfonate), Lysinoalanine, Syntheticlysinoalanine (N-ε-DL-(2-amino-2-carboxyethyl)-L-lysine), tetracycline,alpha lipoic acid (ALA), Dimercaptosuccinic acid, (DMSA),2,3-Dimercapto-1-propanesulfonic acid (DMPS), Calcium disodium versante(CaNa₂-EDTA), D-penicillamine, Deferoxamine, Defarasirox, Dimercaprol(BAL), the calcium salt of diethylene triamine pentaacetic acid (DTPA),or any other metal chelator known in the art. In a preferrednon-limiting embodiment, the metal chelator is pyrrolidinedithiocarbamate (PDTC). The composition of the invention may comprisethe metal chelator in a concentration of between about 0.01 and 10mg/ml, more preferably between about 0.5 and 5 mg/ml, more preferablybetween about 1 and 2 mg/ml, and most preferably about 1.5 mg/ml.

In a further non-limiting embodiments, the plant extract may be derivedfrom, for example but not limited to, Allium sativum (garlic), Ginkgobiloba, tomato, orchid, guava, ginseng, for example Pfaffia paniculata(Brazilian ginseng); Zingiber officinale (ginger); or tobacco, whereinthe orchid is preferably of the genus Cymbidium, for example, yellow orgreen orchids from the genus Cymbidium (Cymbidium ssp). Alternatively,the orchid may be of the genus Dendrobium, for example, Dendrobiumnobile or Dendrobium moschatum.

The extract from plants may be obtained by adding a solvent, such as,for example, alcohol, to the plant tissue, for example, but not limitedto, roots, cloves, flower petals, or leaves which may be chopped, ormacerated prior to mixture with the solvent. The solvent may be mixedwith the plant tissue in a proportion of between 1:99 and 60:40, morepreferably between 15:85 and 50:50 and most preferably between30-40:70-60 of plant mass:alcohol. The solvent can be an alcohol, forexample, ethanol, methanol, or grain alcohol, and can have aconcentration of between 60% and 100%, more preferably between 70% and95%, and most preferably 92% alcohol. The plant/alcohol mixture may beaged in a dark, anaerobic environment for a period of time between 15days and 24 months, more preferably between 1 and 15 months, and mostpreferably 12 months.

According to the invention, the extract derived from plant comprisesparticles containing nucleic acid (DNA or RNA), wherein the particle isan archaea (preferably non-pathogenic) and/or a nanoarchaea, and furtherwherein the particle is present in an amount effective to prevent orinhibit the growth of a mycoplasma and one or more non-mycoplasmamicroorganisms. Aging of the plant/alcohol mixture increases theconcentration of particles in the mixture.

The plant/alcohol mixture may be purified, and the concentration ofnanoparticles may be increased through one or more filtrations. Themixture may be filtered through pores of between 0.5 μm and 50 μm, morepreferably between 5 μm and 20 μm, and most preferably 11 μm., forexample, but not limited to Whatman qualitative filter paper grade 1,diameter 24 cm, pore size 11 μM. Vacuum chambers can also be usedseparately, or in addition to other filtration methods. Additionally,glass microfiber filters may be used for filtration, for example, butnot limited to, a 47 mm diameter glass microfiber filter with a poresize of 1.1 μm. Any filtration methods known in the art may be used tofilter the aged plant/alcohol mixture.

In a non-limiting embodiment, the plant/alcohol mixture can be subjectedto additional aging during the filtration process. For example, oliveoil may be added to the filtrate to create a 1% olive oil filtratemixture, followed by an additional month of storage in a dark anaerobicenvironment.

According to the methods of the present invention, aging a plant extractincreases the proportion of non-pathogenic archaea to pathogenic archaeain the plant extract.

In one embodiment, aging the plant extract increases the number ofnon-pathogenic archaea in the plant extract.

In another embodiment, aging the plant extract decreases the number ofpathogenic archaea in the plant extract.

In another embodiment, an aged plant extract, or alternatively, a plantextract that has not been aged, can be diluted with a dilutant and agedfor an additional period of time.

In a further non-limiting embodiment, the dilutant can be thermal water,oil, for example, olive oil, or any other dilutant known in the art.

In another non-limiting embodiment, the plant extract or the dilutedplant extract can be aged for between 15 days and 24 months.

In another non-limiting embodiment, the plant extract or the dilutedplant extract, can be aged for 30 days.

Furthermore, the composition may comprise particles and/or nanoparticlescontaining DNA or RNA, wherein the particles are a non-pathogenicarchaea and/or a nanoarchaea, and further wherein the particle ispresent in amounts effective to prevent or inhibit the growth of amycoplasma and one or more non-mycoplasma microorganisms. Thenanoparticles may be between 5-500 nm, more preferably between 15-250nm, and most preferably between 30-150 nm in diameter. Alternatively,the composition may comprise medium particles of between 500 nm and 1.1μm in diameter. Additionally, the compositions may comprise one or acombination of both small and medium particles. The size of a particlecan enlarge or decrease depending on the concentration of water and ionsin a solution comprising the particles, such as, for example, Na+ orCa+.

According to the invention, the purity of the plant extract may bedetermined by microscopic examination of the filtered, aged, plantextract, as described in U.S. Patent Application Publication No.20050142116. For example, the filtered, aged plant extract can bestained with any DNA or RNA dye known in the art, such as acridineorange, bisbenzimide H 33342 (Hoechst), or4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI); and viewed withan immunofluorescence optical microscope, an electron microscope, or anyother microscope known in the art. Two forms of archaea, havingdifferent morphological characteristics may be identified. One typecomprising an electron-dense content may be between about 0.03-0.15 μm(nanoparticle) and about 0.5-1.1 μm in diameter (medium particle) (FIGS.4 and 5, respectively). A second type may comprises a clear, emptycontent, and may be about 0.15-2.4 μm in diameter (FIGS. 5 and 6). Theclear, empty archaea are similar in morphology to the pathogenic archaeaassociated with lesions, while the electron dense archaea comprise thenon-pathogenic archaea and nanoarchaea comprising DNA or RNA. Brilliantred particles, which may comprise metallic ions, may also adhere to thesurface of the archaea. Optimum purity may be achieved whenpredominantly, preferably essentially, only fast moving electron-densenanoparticles are visible. The presence of clear, empty archaea or largebrilliant red particles of about 0.15-0.24 μm and at a concentration of,for example, ≧1.0 large brilliant red particle/visual field, indicatessuboptimal purity. In cases of suboptimal purity, the filtered agedplant extract is subjected to additional filtration, for example,tangential flow filtration in the Minitan Ultrafiltration System(Millipore, Bedford, Mass., USA), using the microporous membrane packet(30,000 NMWL). In preferred embodiments, the compositions of theinvention comprise a greater number of electron dense archaea(nanoparticles and medium particles) than empty, clear archaea; and agreater number of archaea not associated with large brilliant redparticles than those associated with large brilliant red particles.

According to the invention, the purified plant extract may comprise anenriched population of particles. The concentration of particles may bebetween 1×10⁵ and 1×10¹⁰ particles/ml, more preferably between 1×10⁶ and1×10⁹ particles/ml, and most preferably about 1×10⁷ particles/ml.

In a non-limiting embodiment, the compositions of the invention comprisecombinations of trans-sialidase, a metal chelator, and one or morepurified plant extracts as shown in Table I.

TABLE I Combinations of trans-sialidase, a metal chelator, and one ormore purified plant extracts encompassed by the invention. Combinationsof trans-sialidase (TS), pyrrolidine dithiocarbamate (PDTC), andpurified plant extracts TS TS + PDTC TS + PDTC + Allium sativum (AS)TS + PDTC + Ginko biloba (GB) TS + PDTC + Zingiber officinale (ZO) TS +PDTC + orchid extract (OE) TS + PDTC + AS + GB TS + PDTC + AS + ZO TS +PDTC + AS + OE TS + PDTC + AS + GB + ZO TS + PDTC + AS + GB + OE TS +PDTC + AS + GB + ZO + OE TS + PDTC + AS + ZO + OE TS + PDTC + GB + ZOTS + PDTC + GB + OE TS + PDTC + GB + ZO + OE TS + PDTC + ZO + OE TS + ASTS + GB TS + ZO TS + OE TS + AS + GB TS + AS + ZO TS + AS + OE TS + AS +GB + ZO TS + AS + GB + OE TS + AS + GB + ZO + OE TS + AS + ZO + OE TS +GB + ZO TS + GB + OE TS + GB + ZO + OE TS + ZO + OE

5.2 Therapeutic Uses

The present invention provides for compositions and methods for reducingthe presence of atherosclerotic plaques in a blood vessel. In apreferred embodiment, the composition of the invention comprises atrans-sialidase enzyme, PDTC, and one or more purified plant extractsThe compositions and methods of the invention further provide forreducing the level of total serum cholesterol in a treated subject, aswell as serum LDL, HDL and triglyceride levels.

In one embodiment, the composition of the invention may be administeredin an amount effective to reduce the presence of an atheroscleroticplaque. In a non-limiting embodiment of the invention, the compositionmay be administered systemically, for example, as an injection. Inanother preferred embodiment of the invention, the composition may beadministered orally. According to the invention, the composition iseffective to promote a reduction in the presence of one or moremycoplasma and one or more non-mycoplasma microorganism on a bloodvessel wall as compared to a subject not treated with the composition.For example, the presence of Mycoplasma pneumoniae and Chlamydiapneumoniae is reduced in atherosclerotic plaques.

In another series of non-limiting embodiments, the composition may beadministered as a single dose, or at regular intervals so that thecomposition is effective to promote a reduction in the presence or levelof atherosclerotic plaques, total serum cholesterol, serum LDL, serumHDL, and triglyceride in a subject as compared to a subject not treatedwith the composition.

In a non-limiting embodiment of the invention, the composition may beadministered in an amount effective to reduce the surface area of ablood vessel covered by an atherosclerotic plaque. The composition maydecrease the percentage of a blood vessel's surface area occupied by aplaque to between about 0% and 75%, more preferable between 2% and 50%,more preferably between 5% and 60%, more preferably between 10% and 25%and most preferably about 11% of the total surface area of the bloodvessel.

In another non-limiting embodiment of the invention, the composition maybe administered in an amount effective to reduce the level of totalserum cholesterol in a subject in need of treatment. The composition mayreduce the level of total serum cholesterol of the subject by about 5%,10%, 20%, 50%, 90% or 95% such that the level of total cholesterol isreduced to about the normal level found in a subject not in need oftreatment.

In another non-limiting embodiment of the invention, the composition maybe administered in an amount effective to reduce the level of serum LDLcholesterol in a subject in need of treatment. The composition mayreduce the level of serum LDL cholesterol of the subject by about 5%,10%, 20%, 50%, 90% or 95% such that the level of serum LDL cholesterolis reduced to about the normal level found in a subject not in need oftreatment.

In another non-limiting embodiment of the invention, the composition maybe administered in an amount effective to reduce the level of serum HDLcholesterol in a subject in need of treatment. The composition mayreduce the level of serum HDL cholesterol of the subject by about 5%,10%, 20%, 50%, 90% or 95% such that the level of serum HDL cholesterolis reduced to about the normal level found in a subject not in need oftreatment.

In another non-limiting embodiment of the invention, the composition maybe administered in an amount effective to reduce the level oftriglycerides in a subject in need of treatment. The composition mayreduce the level of triglycerides of the subject by about 5%, 10%, 20%,50%, 90% or 95% such that the level of triglycerides is reduced to aboutthe normal level found in a subject not in need of treatment.

In a further non-limiting embodiment of the invention, the normal levelof total serum cholesterol is about 200 mg/dl or less, the normal levelof serum LDL cholesterol is about 100 mg/dl or less, the normal level ofserum HDL cholesterol is about 60 mg/dl or more, and the normal level oftriglycerides is about 150 mg/dl or less (American Heart Associationwebsite, Jan. 30, 2007).

In another non-limiting embodiment of the invention, the composition maybe administered in an amount effective to reduce the presence of one ormore microorganism with an atherosclerotic plaque, for example, but notlimited to Mycoplasma pneumoniae and Chlamydia pneumoniae, wherein thereduction in microorganism presence is indicated by a reduction in thedetection of the microorganisms' antigens. According to the invention,the reduction in antigen detection is between about 0.1 and 100%, andmost preferably 99% as compared to the antigen detection in an untreatedsubject.

The composition may be administered locally or systemically, forexample, by injection, orally, occularly, rectally, topically, or by anyother means known in the art. The composition may be ingested as aliquid, a pill, or a capsule (e.g. liquid or powder-filled).

In one non-limiting embodiment, the composition may comprise atrans-sialidase, a metal chelator, for example, but not limited to,PDTC, NTA, diphenylthiocarbazone(dithizone), histidine, DP-109, EGTA,EDTA, DMPS, Lysinoalanine, Synthetic lysinoalanine, tetracycline, ALA,Dimercaptosuccinic acid, DMSA, Calcium disodium versante,D-penicillamine, Deferoxamine, Defarasirox, Dimercaprol, and DTPA; andone or more purified plant extract. The trans-sialidase may have anenzymatic activity of between about 0.01 and 10 U/ml, more preferablybetween about 0.2 and 5 U/ml, more preferably between about 0.5 and 2U/ml and most preferably about 1.0 U/ml. The metal chelator may have aconcentration of between about 0.01 and 10 mg/ml, more preferablybetween about 0.5 and 5 mg/ml, more preferably between about 1 and 2mg/ml, and most preferably 1.5 mg/ml. The purified plant extract maycomprise a particle concentration of between about 1×10⁵ and 1×10⁷particles/ml, more preferably between about 5×10⁶ and 9×10⁶particles/ml, more preferably between about 2×10⁶ and 3×10⁶particles/ml, and most preferably about 1.0×10⁶ particles/ml.

In a specific non-limiting embodiment, the composition is administeredas an injection, wherein the composition comprises a trans-sialidase,PDTC and one or more purified plant extract, further wherein thetrans-sialidase has an enzymatic activity of 1.04 U/ml, the PDTC has aconcentration of 1.5 mg/ml, and the purified plant extract has aparticle concentration of 1.0×10⁶ particles/ml.

In an alternative non-limiting embodiment, the composition may comprisea trans-sialidase, a metal chelator, and one or more purified plantextract, wherein the trans-sialidase comprises an enzymatic activity ofbetween about 1×10⁻⁸ and 1×10⁻⁴ U/ml, more preferably between about1×10⁻⁷ and 1×10⁻⁵ U/ml, more preferably between about 1×10⁻⁶ and 5×10⁻⁶U/ml and most preferably about 1.5×10⁻⁶ U/ml. The metal chelator mayhave a concentration of between about 0.01 and 10 mg/ml, more preferablybetween about 0.5 and 5 mg/ml, more preferably between about 1 and 2mg/ml, and most preferably 1.5 mg/ml. The purified plant extract maycomprise a particle concentration of between about 1×10⁵ and 1×10⁷particles/ml, more preferably between about 2×10⁶ and 9×10⁶particles/ml, more preferably between about 3×10⁶ and 7×10⁶particles/ml, and most preferably about 5×10⁶ particles/ml.

In a specific non-limiting embodiment, the composition is administeredorally as a liquid, wherein the composition comprises a trans-sialidaseand one or more purified plant extract, further wherein thetrans-sialidase has an enzymatic activity of 1.3×10⁻⁶ U/ml and thepurified plant extract has a particle concentration of 5.0×10⁶particles/ml.

In another non-limiting embodiment, the composition is administered inan amount of between 0.002 and 5.0 ml/kg, more preferably between 0.1and 2.0 ml/kg, more preferably between 0.2 and 1.0 ml/kg, and mostpreferably about 0.25-0.5 ml/kg.

In a further non-limiting embodiment, the composition may beadministered once, twice, three, four, five, or six or more times perday during the treatment period. Alternatively, the composition may beadministered once every two, three, four, five, six or seven or moredays.

In a non-limiting example of the invention, the composition is a mixtureof trans-sialidase and PDTC, wherein the trans-sialidase has an activityof about 1.04 U/ml and the PDTC is at a concentration of 1.5 mg/ml, andwherein the composition is administered via intraperitoneal orintravenous injection at a volume of about 25-0.5 ml/kg every other day.

In a further non-limiting example of the invention, the mixture oftrans-sialidase and PDTC is supplemented with a purified plant extractdiluted 1:10 in purified water, and containing an average of 1.0×10⁶nanoparticles/ml. The plant extract dilution is administered throughintraperitoneal injections once per day for a four week treatmentperiod. Examples of mixtures include, but are not limited to, TS+PDTC,TS+PDTC+AS extract, TS+PDTC+AS+GB extracts, and TS+PDTC+AS+GB+ZOextracts. For each of the mixtures, the TS+PDTC may be injectedintravenously or ingested orally in an amount of 0.25-0.5 ml/kg everyother day during a 12 week treatment session, wherein the mixturecomprises 1.04 U/ml TS activity and 1.5 mg/ml PDTC. Each of the plantextracts comprise a 1:10 plant extract:water dilution which furthercomprise 1.0×10⁶ nanoparticles/ml. A total volume of 1 ml of dilutedplant extract is injected intraperitoneally once daily during the 12week treatment period. When more than one diluted plant extract is used,the different extracts are mixed in equal volumes.

6 EXAMPLES Example 1 Treatment of Aortic Atherosclerotic Plaques inRabbits with Trans-Sialidase, PDTC, and Plant Extracts

The present study compares the effects of trans-sialidase (TS) enzymederived from Trypanosoma cruzi, PDTC and one or more aged plant extractsderived from Allium sativum (AS), Ginkgo biloba (GB) and Zingiberofficinale (ZO), on the reduction of aortic atherosclerotic plaques,lipid serum levels and infectious agent antigens at intima in rabbitsreceiving cholesterol-rich-diet.

Material and Methods

White New Zealand male rabbits of approximately 2 months in age,weighing 2.2±0.5 kg were included in the study. The study lasted 12weeks. The rabbits were divided into six different treatment groups.Group I animals received normal rabbit chow, while Groups II-VI receivednormal rabbit chow supplemented with 1% cholesterol. Animals receivedthe diets for a period of 12 weeks. Groups III-VI also receivedanti-atherosclerotic treatment during the final 4 weeks of the study.The feeding and treatment schedule is shown in Table II.

TABLE II Feeding and treatment schedule for the six study Groups. Numberof Anti-Atherosclerotic Rabbits Treatment (final 4 Group in Group Dietweeks of study) GI. 13 Normal rabbit chow None GII. 13 Normal rabbitchow + None 1% cholesterol GIII. 5 Normal rabbit chow + TS + PDTC 1%cholesterol GIV. 5 Normal rabbit chow + TS + PDTC + AS 1% cholesterolextract GV. 5 Normal rabbit chow + TS + PDTC + AS + 1% cholesterol GBextracts GVI. 5 Normal rabbit chow + TS + PDTC + AS + 1% cholesterolGB + ZO extracts

Diet Preparation

Nuvilab® (Nuvital. Curitiba, PR. Brazil) was used as the normal rabbitchow in the study. Normal rabbit chow supplemented with 1% cholesterolwas prepared by adding 10 g of cholesterol powder (Sigma—C 8503)dissolved in a solution of 50 ml ethylic ether and 100 ml 70% ethanol,to each Kg of normal rabbit chow

Trans-Sialidase (TS) Preparation

Trypanosoma cruzi were cultured according to Kloetzel et al. (Kloetzelet al., 1984, Trypanosoma cruzi interaction with macrophages:differences between tissue culture and bloodstream forms. Rev. Inst.Med. Trop. Sao Paulo., 26:179-85). Supernatant from the culture wasfiltered through a 1 μm pore filter in a vacuum chamber, or thesupernatant was filtered through a 0.22 μm filter and concentrated byprecipitation with 50% (NH₄)₂SO₄. The precipitates were dialyzed againstphosphate-buffered saline, and then passed through a tresyl-agarosecolumn containing an immobilized anti-trans-sialidase monoclonalantibody. The column was washed with phosphate-buffered saline, followedby a 10 mM sodium phosphate, pH 6.5 wash. The trans-sialidase was elutedwith a 3.5 mM MgCl₂, 10 mM sodium phosphate, pH 6.0 solution. Thefractions eluted from the column were immediately filtered through aSephadex G-25 column equilibrated with 20 mM Tris-HCl, pH 8.0, to removeMgCl₂. The trans-sialidase was further purified by passage through aMono Q column equilibrated in 20 mM Tris-HCl, pH 8.0, and eluted with alinear gradient from 0 to 1 m NaCl in the same buffer.

The purified enzyme derived from the culture comprises a 400 kDamultimeric aggregate. The enzymatic activity of the purifiedtrans-sialidase was measured according to methods described inInternational Patent Application No. PCT/BR01/00083, filed Jul. 3, 2001.Purified trans-sialidase used in the study had an enzymatic activity of1.3 U/ml.

Plant Extract Preparation

Plant (Allium sativum (AS) cloves, Ginkgo biloba leaves (GB) andZingiber officinale (ZO) raw) extracts were prepared by introducingsliced plant tissue into a 10-20% aqueous ethanol solution. Theplant/ethanol mixture was adjusted to a final proportion of 40:60 plantweight:ethanol and stored for up to 12 months at room temperature in adark, anaerobic environment (in a sealed bottle). Following storage, theplant mass/alcohol mixture was passed through Whatman qualitative filterpaper grade 1, diameter 24 cm, pore size 11 μm. The liquid filtrate wasthen filtered again in a vacuum chamber with a 47 mm diameter glassmicrofiber filter, pore size 1.1 μm. Then filtrate was next filteredthrough successively smaller pores, in a tangential flow device (MinitanUltrafiltration Millipore System—Millipore, Bedford, Mass., USA), usingthe microporous membrane packet (30,000 NMWL) that concentrates largeparticles. The filtrated portion of the extract was used in the study.

Trans-Sialidase (TS)+PDTC Anti-Atherosclerotic Treatment

Rabbits were treated with 0.25-0.5 ml/kg of a trans-sialidase+PDTCmixture injected intraperitoneally on alternative days. 1 ml of thetreatment mixture comprised 0.8 ml of Trypanosoma cruzi culturesupernatant (enzymatic activity of 1.3 U/ml) and 1.5 mg of PDTC(pyrrolidine dithiocarbamate ammonium salt from ICN Biomedicals Inc.,Aurora, Ohio, USA.) dissolved in 0.2 ml of saline.

Trans-Sialidase (TS)+PDTC+Plant Extract Anti-Atherosclerotic Treatment

Animals were treated with the trans-sialidase+PDTC solution as describedabove along with 1 ml of a purified plant extract dilution containing anaverage of 1×10⁶ nanoparticles. The plant extract dilution wasadministered through intraperitoneal injections once per day during thefour week treatment period. The purified plant extract dilution wasgenerated by diluting an aged ethanolic plant extract 1:10 in water.

Serum Lipid Analysis

Serum lipid analysis was performed at the beginning and end of the 12week experiment. To obtain the blood serum, a 10 ml blood sample wastaken from each animal through cardiac puncture, and centrifuged at 1500g for 15 min at 4° C. Total cholesterol, high-density lipoprotein (HDL)and triglycerides concentrations were determined by enzymatic methods(CHOD-PAP Merck®, USA. and GPO-PAP Cobas Mira, Roche®).

Aortic Atherosclerotic Lesions Analysis

To analyze aortic atherosclerotic lesions, rabbits were euthanized withan intramuscular injection of 25 mg/kg Ketamine and 2-5 mg/kg Xilazina.Aorta were excised and opened longitudinally along the anterior wall,washed in saline, stretched on cardboard, and placed in 10% bufferedformalin. Aorta were then stained with Sudan IV. Intimal positive areasstained in red by Sudan IV were measured by automatic detection using animage analysis system (Quantimet 500, Leica).

Histological examination of the aorta were also performed. A 1 cmthickness cross-section of the initial descending thoracic aorta weretaken and embedded in paraffin. 5 μm serial sections of thecross-section were submitted to H&E stain and immunohistochemicaldetection of Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP)antigens, as previously described. (Fagundes RQ. Study ofco-participation of natural infection by Chlamydophila pneumoniae andMycoplasma pneumoniae in experimental atherogenesis in rabbits. Doctoralthesis presented at the Heart Institute of Clinical Hospital, in theCardiology Sciences Post graduation Program of Sao Paulo UniversitySchool of Medicine, Mar. 17, 2006). The percentage of area positive forinfectious agent antigens on the immunostained slides was determinedusing an automatic color detection system (Image Analysis SystemQuantimet 500, Leica, Germany).

Results

The mean and standard deviation values of percentage areas of fatplaques (macroscopically) and of MP and CP antigens at intima, andintimal area in 1 cm cross section are shown at table III. Lipid levelsin the serum are reported at table IV.

Atherosclerotic Plaques and Lipid Levels

The control group, Group I, which received normal rabbit chow and noant-atherosclerotic treatment, did not develop plaques on the aortalwalls. Trace amounts of MP and CP antigens on the aorta wall weredetected, but in all cases, without development of atheroma plaques.

Group II, which received normal food supplemented with 1% cholesteroland no anti-atherosclerotic treatment, presented 75% coverage of theaorta intimal surface by severe lipid atheroma plaques stained withSudan IV. (FIG. 1). The histology revealed that the plaques werecomprised of 89% fat.

Group III, which received normal food supplemented with 1% cholesteroland treatment with TS+PDTC, exhibited 50% coverage of the aorta intimalsurface by severe lipid atheroma plaques stained with Sudan IV. (FIG.1).

Groups IV, V and VI, which received normal food supplemented with 1%cholesterol and treatment with TS+PDTC+Plant extracts, presentedprogressively smaller areas of atherosclerotic plaque coverage of theaorta wall (Table III). The addition of AS to the treatment regimereduced the levels of total cholesterol and HDL in the blood serum(Table IV), but did not reduce the % plaque area of atheroma (TableIII), and induced a decrease in aorta perimeter, indicating a negativeremodeling of the vessel. The addition of AS+GB to the treatment led toa significant reduction in both % area of intimal plaques andcholesterol levels in the serum. The most effective anti-atheroscleroticeffect was observed with a complex of plant extracts from AS, GB and ZO,which reduced the area of the aorta wall covered by plaque to 11%, andreturned lipid levels in the serum to normal levels (Table IV). Most ofthe remaining intimal plaques were fibrotic, largely free of foam cells(FIG. 1). Treatment with AS, GB and ZO extracts reduced both intimalarea and % of intraplaque fat (Table III).

TABLE III Intimal Area and Percentage Areas of Aorta Atheroma Plaques,Fat and Infectious Agents in Aortic Plaques of 1% Cholesterol-FedRabbits Submitted to Different Treatments. Intima % area % area % Plaquearea - area C. pneumoniae+ M. pneumoniae+ macroscopic % plaque fat (mm²)Group Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) GI 0.007 (0.005)0.013 (0.012) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) GII 23.50 (5.66) 25.60(3.96) 75 (9) 89 (5) 75 (7) GIII 16.04 (0.60) 12.81 (1.27) 50 (3) 50 (3)65 (4) GIV 12.60 (0.85) 10.53 (0.51) 67 (14) 61 (10) 61 (8) GV 8.60(0.21) 4.57 (0.51) 42 (8) 40 (14) 39 (6) GVI 0.022 (0.005) 0.02 (0.005)11 (1) 17 (10) 17 (2)

TABLE IV Cholesterol Fractions and Triglycerides Serum Levels of 1%Cholesterol-Fed Rabbits Submitted to Different Treatments. Values shownare in mg/dl. Total cholesterol Triglycerides HDL LDL Mean Mean MeanMean Group (SD) p (SD) p (SD) p (SD) p GI 67 (31) 0.17 64 (13) 0.01 42(7) <0.01 33 (24) 0.09 GII 1029 (237) <0.01 115 (55) <0.01 210 (52)<0.01 775 (227) <0.01 GIII 873 (82) 0.09 95 (10) 0.22 175 (17) 0.08 743(92) 0.38 GIV 778 (58) 0.02 86 (9) 0.13 115 (11) <0.01 635 (60) 0.10 GV408 (69) <0.01 51 (6) <0.01 90 (6) <0.01 335 (29) <0.01 GVI 53 (6) <0.0147 (8) 0.26 36 (3) <0.01 18 (6) <0.01 GI - control group not receivingcholesterol diet; GII - non treated; GIII- received trans-sialidase (TS)and pyrrolidine dithiocarbamate (PDTC); GIV - received TS + PDTC +Allium Sativum extract (AS); GV - treated with TS + PDTC + AS + Ginkgobiloba extract (GB) and GVI - received TS + PDTC + AS + GB + Zingiberofficinale extract; SD—standard deviation p - represents differenceregarding the respective values of the above group, except GI valueswhich were compared with group GVI (level of significance 5%)Mycoplasma pneumoniae and Chlamydia pneumoniae Antigens

Treatment with TS+PDTC (Group III) reduced the percent area of MPantigen expression from 25.6±3.96 to 12.81±1.27 (p<0.01) and CP antigenexpression from 23.50±5.66 to 16.04±0.60 (p<0.001) as compared to GroupII animals that received no anti-atherosclerotic treatment. Addition ofplant extracts caused a progressively more significant decrease inpercentage area positive for CP and MP antigens at intima. When allthree plant extracts were used in the anti-atherosclerotic treatment,the reduction was more effective. Use of all three extracts reduced thepercentage of total area expressing MP antigen to 0.02±0.005 and CPantigens to 0.022±0.005. These values were similar to the control group(Group I) (Table III). Macroscopic and microscopic aspects of differentgroups are exemplified at the FIG. 1.

CONCLUSION

In conclusion, the present study indicates a new formulation for thetreatment of atherosclerosis, using a combination of T. cruzitrans-sialidase, PDTC and three aged plant extracts: Allium sativum,Ginkgo biloba and Zingiber officinale. Treatment with these compoundswas effective in reducing intimal accumulation of both fat and C.pneumoniae plus M. pneumoniae antigens. The lipid serum levels returnedto normal levels even in the permanence of a cholesterol rich diet.

Example 2 Treatment of Human Patients Exhibiting High Total Cholesteroland LDL Levels with Trans-Sialidase and Plant Extracts

Three volunteers who presented high levels of total cholesterol and LDLcholesterol fraction in blood serum were treated with purifiedtrans-sialidase and aged plant extracts.

Materials and Methods

Plant Extract Preparation

Plant (Allium sativum (AS) cloves, Ginkgo biloba leaves (GB). Zingiberofficinale raws (ZO) and Pfaffia paniculata (Brazilian ginseng) roofs(GS)) extracts were prepared by introducing sliced plant tissue into a10-20% aqueous ethanol solution. The plant/ethanol mixture was adjustedto a final proportion of 40:60 plant weight:ethanol and stored for up to12 months at room temperature in a dark, anaerobic environment (in asealed bottle). Following storage, the plant mass/alcohol mixture waspassed through Whatman qualitative filter paper grade 1, diameter 24 cm,pore size 11 μm. The liquid filtrate was then filtered again in a vacuumchamber with a 47 mm diameter glass microfiber filter, pore size 1.1 μm.Then filtrate was next filtered through successively smaller pores, in atangential flow device (Minitan Ultrafiltration Millipore System,Millipore, Bedford, Mass., USA), using the microporous membrane packet(30,000 NMWL) that concentrates large particles. The filtrated portionof the extract was used in the experiments.

Recombinant Trans-Sialidase Purification

Recombinant trans-sialidase was produced and purified from theEscherichia coli strain BLB21 DE3 inserted with a pTSII plasmodiumcomprising the T. cruzi trans-sialidase gene as described inInternational Patent Publication WO/2002/002050 by Higuchi et al.,published Jan. 10, 2002.

The protein concentration of 5 mg/ml was produced as measured with aspectrophotometer. The recombinant trans-sialidase was diluted in abuffer liquid (TBS+BSA 0.2%), and the activity was measured according topreviously described methods (International Patent PublicationWO/2002/002050). The purified enzyme was diluted 1:10,000 and 1:100,000resulting in enzymatic activities of 15,000 and 5,000 CPM, respectively.For human oral administration, the trans-sialidase was diluted1:1,000,000 (0.005 mg/ml) in MilliQ purified water, and stored at 4° C.

Preparation and Administration of Oral Drug

Equal proportions of pure extracts from Allium sativum (AS); Ginkgobiloba (GB) Zingiber officinale (ZO) and ginseng (GS) were mixed. Themixture was then diluted 1:1 in thermal water (from Irai, RS, Brazil),which was previously boiled and filtered.

Trans-sialidase diluted 1:1,000,000 (0.005 mg/ml) was administered tothe subjects. A mean of 200 ul to 500 ul (4-10 drops) of dilutedtrans-sialidase was added in a glass of water and ingested daily.

Three volunteers who presented high levels of total cholesterol andserum LDL cholesterol were treated with the oral compositions for aminimum of 30 days to over one year. The volunteers were administeredorally 200 ul of the diluted plant extract composition 2×/day, and 200ul of the diluted trans-sialidase composition 1×/day. The patients werealso being treated with other anti-cholesterol drugs (statins).Following treatment, the volunteers presented normal total cholesteroland serum LDL cholesterol levels, wherein the mean level of decrease inserum cholesterol levels following treatment was 20%. This decrease wasobserved even if statins had been previously used to lower serumcholesterol levels.

Example 3 Tobacco Extracts Contain Large Lipidic Pathogenic Archaea thatcan be Removed NY Incubation in Thermal Water Tobacco Extracts:

Tobacco extracts were obtained by removing the contents from a packet ofcommercial tobacco cigarettes, and adding the contents to 80 ml ofwater. The tobacco/water mixture was then mixed with 500 ml of ethanol(92% ethanol). The tobacco/water/alcohol mixture was then aged in asealed bottle for 12 months. Following 12 moths of aging, the mixturewas filtered through Whatman qualitative filter paper (grade 1, diameter24 cm, pore size 11 μm). The filtrate was then filtered a second timethrough vacuum chambers comprising a 47 mm diameter glass microfiberfilter with a pore size of 1.1 μm.

The filtrate was analyzed with fluorescent and electron microscopy asdescribed in U.S. Patent Application Publication No. 20050142116.Fluorescent microscopy of filtrate stained with acridine orange showed alarge number of both large particles and nanoparticles containing DNA orRNA, but the filtrate was predominated by the large particles.

Analysis of the filtrate with electron microscopy showed that the twoparticles were the two types of archaea described previously: very smalland clear structures of about 0.03-0.15 μg/m in diameter (see FIG. 4),which correspond to non-pathogenic archaea; and large particles(0.15-0.24 μm), along with other electron dense lipidic structures,which correspond to pathogenic archaea (see FIG. 5). The large archaeaparticles may also be observed as round brilliant red particles underfluorescent microscopy.

The pathogenic large particle archaea are also found in humanperiadventitial adipose tissue of atherosclerotic aortic aneurysmsAnalysis of human periadventitial adipose tissue of atheroscleroticaortic aneurysms with electron microscopy showed that this tissuecontained a large number of the large lipidic particles surrounded byinflammatory infiltrate archaea surrounded by inflammatory lymphocytes(FIG. 6) suggesting that the particles are recognized as foreignstructures by the immune system. High magnification of theses lipidicparticles (FIG. 7) shows that the particles contain a clear externalmembrane, indicating that these particles correspond to microbes (largelipidic archaea), and not to lipidic droplets in the cytoplasm. Theselipidic large archaea have the same morphology as the large particlesthat predominate tobacco extract, and as shown in FIGS. 5 and 6.

Preparation of the Therapeutic Extract from Tobacco:

As described previously, diluting and aging plant ethanolic extractsresults in an extract enriched with non-pathogenic archaes (see U.S.Patent Application Publication No. 20050142116). For example, dilutingan ethanolic plant extract with thermal medicinal water (from Irai cityin South of Brazil) in a proportion of 1:10 (extract/water), and agingthe mixture for 30 days, results in a reduction of the large lipidicarchaea particles, while retaining the small non-pathogenic archaea.Extracts with enriched non-pathogenic archaea have been shown to beuseful in the treatment of atherosclerosis and lowering serum lipids.Accordingly, tobacco extract prepared as described above and aged for 12months was diluted 1:10 in thermal water, and aged for an additional 30days.

Atherosclerosis was induced in a rabbit by feeding the rabbit with ahigh cholesterol diet (5% cholesterol) for 12 weeks. Following the 8weeks of the feeding period, 0.5 ml samples of the aged 1:10 tobaccoextract/thermal water mixture (which was enriched with non-pathogenicarchaea) was subcutaneously injected into the rabbit's ear, twice aweek, during the last 4 weeks of cholesterol enriched diet program. Theanimal was then sacrificed followed by macroscopic and microscopicanalysis of the ascending and descending thoracic aorta. Both analysesdid not show any atheroma plaques in the ascending or descendingthoracic aorta, which are normally present following a cholesterolenriched feeding program (see FIG. 1).

CONCLUSION

The use of thermal medicinal water to dilute aged ethanolic plantextracts is effective in eliminating undesirable pathogenic largeparticle archaea, and preserving non-pathogenic archaea present in theextracts. Such an observation is observable by direct visualization ofthe plant extract mixture with fluorescent microscopy before and afterdiluting the extract with thermal water. Thus, the use of thermal waterto purify plant extracts may increase the therapeutic and medicinalproperties of the extracts. For example, non-pathogenic archaea presentin tobacco extract may be enriched through purification with thermalwater, and used to treat cholesterol induced atherosclerosis. Onehypothetical mechanism of the success of such a treatment is that inhuman atherosclerotic lesions, such as aneurysms or unstable plaquesthat cause myocardial infarction, there are higher numbers of pathogenicarchaea. These pathogenic arachae in the lesions may be increased by theuse of tobacco products. Surprisingly, increasing the non-pathogenicarchaea present in tobacco extracts by diluting the extracts withthermal water, may enable tobacco to be used as a treatment to combatthe pathogenic archaea and atherosclerosis.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1. A method of treating a disorder characterized by undesirable cell proliferation in a subject by administering a composition to the subject in an amount effective to inhibit the undesirable cell proliferation or prevent the disorder associated with the undesirable cell proliferation, wherein the composition comprises an agent that can remove sialic acid residues and one or more plant extract comprising nucleic acid-containing particles selected from the group consisting of archaea, nanoarchaea, and a mixture thereof.
 2. The method of claim 1, wherein the composition further comprises a metal chelator.
 3. The method of claim 1, wherein the disorder is selected from the group consisting of atherosclerosis, elevated total serum cholesterol, elevated serum low density lipoprotein, elevated serum high density lipoprotein, and elevated triglyceride.
 4. The method of claim 1, wherein the agent that can remove sialic acid residues is an enzyme selected from the group consisting of trans-sialidase, neuraminidase, and a combination of a trans-sialidase and a neuraminidase.
 5. The method of claim 1, wherein the plant extract is an extract from a plant selected from the group consisting of garlic, ginkgo, tomato, orchid, guava, ginseng, ginger, and tobacco.
 6. The method of claim 2, wherein the metal chelator is pyrrolidine dithiocarbamate (PDTC).
 7. A composition for treating or preventing a disorder characterized by undesirable cell proliferation comprising an agent that can remove sialic acid residues and one or more plant extract comprising nucleic acid-containing particles selected from the group consisting of archaea, nanoarchaea, and a mixture thereof.
 8. The composition of claim 7, further comprising a metal chelator.
 9. The composition of claim 7, wherein the agent that can remove sialic acid residues is an enzyme selected from the group consisting of trans-sialidase, neuraminidase, and a combination of a trans-sialidase and a neuraminidase.
 10. The composition of claim 7, wherein the plant extract is an extract from a plant selected from the group consisting of garlic, ginkgo, tomato, orchid, guava, ginseng, ginger, and tobacco.
 11. The composition of claim 8, wherein the metal chelator is pyrrolidine dithiocarbamate (PDTC). 